High HBV antigen levels drive loss of vaccine-induced CD8⁺ T-cell function in carrier mice over time

BACKGROUND AND AIM: Chronic hepatitis B virus (HBV) infection persists due to the dysfunction of virus-specific CD8⁺ T cells. High HBV antigen levels impair the success of therapeutic vaccination. The influence of viral titers on vaccine-induced immunity remains to be fully elucidated. We compared the kinetics of vaccine responses in low- and high-titer HBV carrier mice to identify tipping points and dissect the decisive steps determining the outcome of therapeutic vaccination. METHODS: We established low- and high-titer persistent HBV replication in the livers of immunocompetent C57BL/6J mice using adeno-associated-virus (AAV)-HBV and administered TherVacB, a heterologous protein prime-modified vaccinia Ankara boost vaccine. Serum and liver samples were collected during vaccination and subsequently longitudinally to analyze HBV-specific immune responses and viral parameters. RESULTS: HBV antigen levels before vaccination did not significantly influence the anti-HBs response (p>0.9); however, they had a significant effect on the vaccine-induced CD8 T-cell response. TherVacB induced, activated core-specific CD8⁺ T cells efficiently infiltrated into the liver in low- and high-titer HBV carriers. Low-titer HBV carrier mice achieved sustained viral clearance and memory T-cell formation, whereas high-titer mice exhibited only transient viral reduction, followed by CD8⁺ T-cell dysfunction and failure to clear the infection. The persistence of HBV infection and constant exposure to high antigen levels led to the overexpression of exhaustion markers, such as PD-1 and TOX (p<0.01), as well as the co-stimulatory receptor 4-1BB (p<0.01). Core-specific CD8⁺ T cells remained trapped in the liver, progressively lost their effector function, and were eventually eliminated. CONCLUSIONS: These findings identify critical time points that influence vaccine-induced CD8 T-cell responses, suggesting that early intervention targeting T-cell dysfunction may enhance therapeutic vaccination outcomes in chronic HBV infection. IMPACT AND IMPLICATIONS: This study demonstrates that baseline HBV antigen load critically determines the durability of vaccine-induced CD8⁺ T-cell immunity. While therapeutic vaccination initially elicited liver-infiltrating, HBV-specific CD8⁺ T cells irrespective of the viral titer, only low-titer infection permitted sustained viral clearance and memory T-cell formation. In high-titer HBV carriers, persistent antigen exposure drove progressive T-cell exhaustion and deletion, resulting in transient viral control and treatment failure. Importantly, the data identify a critical time window in which early intervention to prevent or reverse T-cell dysfunction may be decisive. Clinically, these findings support combining therapeutic vaccination with antigen-lowering or immunomodulatory strategies applied early to achieve durable immune control in chronic HBV infection.